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J. Biol. Chem., Vol. 283, Issue 41, 27418-27425, October 10, 2008

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An Obligatory Heterodimer of 14-3-3β and 14-3-3 Is Required for Aldosterone Regulation of the Epithelial Sodium Channel^*

From the Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Increased distal nephron sodium absorption in response to aldosterone involves Nedd4-2 phosphorylation, which blocks its ability to ubiquitylate ENaC and increases apical membrane channel density by reducing its endocytosis. Our prior work (Liang, X., Peters, K. W., Butterworth, M. B., and Frizzell, R. A. (2006) J. Biol. Chem. 281, 16323–16332) showed that aldosterone selectively increased 14-3-3 protein isoform expression and that the association of 14-3-3β with phospho-Nedd4-2 was required for sodium transport stimulation. The knockdown of 14-3-3β alone nearly eliminated the response to aldosterone, despite the expression of other 14-3-3 isoforms in cortical collecting duct (CCD) cells. To further examine this marked effect of 14-3-3β knockdown, we evaluated the hypothesis that phospho-Nedd4-2 binding prefers a heterodimer composed of two different 14-3-3 isoforms. We tested this concept in polarized CCD cells using RNA interference and assays of sodium transport and of the interaction of Nedd4-2 with 14-3-3, a second aldosterone-induced isoform. As observed previously for 14-3-3β knockdown, small interfering RNA-induced reduction of 14-3-3 markedly attenuated aldosterone-stimulated ENaC expression and sodium transport and increased the interaction of Nedd4-2 with ENaC toward prealdosterone levels. After aldosterone induction, 14-3-3β and 14-3-3 were quantitatively co-immunoprecipitated from CCD cell lysates, and the association of both isoforms with Nedd4-2 increased. Finally, the knockdown of either 14-3-3β or 14-3-3 reduced the association of Nedd4-2 with the other isoform. We conclude that the two aldosterone-induced 14-3-3 isoforms, β and , interact with phospho-Nedd4-2 as an obligatory heterodimer, blocking its interaction with ENaC and thereby increasing apical ENaC density and sodium transport.

Received for publication, May 13, 2008 , and in revised form, July 18, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants DK54814 and DK72506. This work was also supported by the Cystic Fibrosis Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, University of Pittsburgh School of Medicine, S362 BST, 3500 Terrace St., Pittsburgh, PA 15261. Tel.: 412-648-9498; Fax: 412-648-8330; E-mail: frizzell{at}pitt.edu.

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